Conjugated linoleic acids (CLAs) refers to a mixture of positional and geometric isomers of linoleic acids, which are unsaturated fatty acids considered essential to the human diet and found preferentially in dairy products and meat. CLAs have generated much interest in the academic and business communities because of its nutritional, therapeutic, and pharmacological properties. There are numerous known CLA compositions, along with various known routes to prepare such compositions. See, e.g., U.S. Pat. No. 6,420,577 (Reaney, et al.); U.S. Pat. No. 6,015,833 (Saebo, et. al.); U.S. Pat. No. 6,160,140 (Bhaggan, et. al.); U.S. Pat. No. 6,034,132 and U.S. Pat. No. 6,019,990 (both to Remmereit, J.); and U.S. Pat. No. 6,225,486 (Saebo, et. al.). CLAs have become biologically and commercially important, as they have been observed to inhibit mutagenesis and to provide unique nutritional value.
Typically, CLAs are a mixture of positional isomers of linoleic acid (C18:2) having conjugated double bonds. The cis-9, trans-11 and trans-10, cis-12 isomers are present in greatest abundance in typical CLA compositions, but it is not absolutely certain which isomers are responsible for the biological and heightened nutritional activity observed. It has been noted from labeled uptake studies that the 9,11 isomer appears to be somewhat preferentially taken up and incorporated into the phospholipid fraction of animal tissues, and to a lesser extent the 10,12 isomer. (See Ha, et al., Cancer Res., 50: 1097 (1991)).
The properties of unsaturated fatty acids and their derivatives can be altered by rearrangement, i.e., isomerization, of the structure of the double bond, either with respect to the steric position or the position in the carbon chain of the molecule of the fatty acid. As noted above, conjugated fatty acid derivatives are of great technical and commercial interest and, therefore, many attempts have been made to isomerize unconjugated fatty acids to conjugated ones. Without being bound by any particular theory, it is believed that such a shifting of the double bond is possible because the conjugated form has a lower state of energy than the unconjugated form.
Previously known routes to produce conjugated unsaturated compounds include hydrogenation of fats using a variety of catalysts. These routes, however, often lead to incomplete isomerization and unwanted side reactions, such as polymerization and intramolecular cyclization. Other known routes include isomerization with an excess of alkali metal hydroxide in an aqueous or alcoholic medium, which leads to a quantitative isomerization. However, this route suffers from the limitation that a considerable excess of alkali metal hydroxide must be used, so that the conjugated fatty acids or fatty acid compounds are obtained in the form of their alkali soaps and have to be recovered and isolated accordingly. These techniques differ in the use of a particular solvent, temperature and pressure. See, e.g., U.S. Pat. No. 3,162,658 (Baltes, et. al.).
The rearrangement of the double bonds of linoleic acids to conjugated positions has been shown to occur during treatment with catalysts such as nickel or alkali at high temperatures, and during auto oxidation. Theoretically, eight possible geometric isomers of 9,11 and 10,12 octadecadienoic acid (c9, c11; c9, t11; t9, c11; t9, t11; c10, c12; c10, t2; t10, c12 and t10, t12) would result from the isomerization of c9, c12-octadecadienoic acid. Again, without being bound by any particular theory, a general mechanism for the isomerization of linoleic acids has been described by J. C. Cowan in JAOCS 72:492-99 (1950). The formation of certain isomers of CLAs is thermodynamically favored as described therein. The relatively higher distribution of 9,11 and 10,12 isomers apparently results from the further stabilization of the c9, t11 or t10, c12 geometric isomers.
U.S. Pat. No. 6,420,577 (Reaney, et al.) describe a process for making CLAs by reacting a linoleic acid-rich oil with a base, in the presence of a catalytic amount of such a base, in an aqueous medium via simultaneous saponification and quantitative isomerization. However, this process utilizes a heightened temperature (>170° C.). Higher temperatures lead to the formation of undesirable CLA isomers, including the trans, trans-CLA isomers.
U.S. Pat. No. 6,160,140 (Bhaggan, et al., the '140 patent) claims the conversion of a linoleic acid-containing oil, free fatty acid or alkyl ester to CLAs by treating it with a base in an alcohol solution, where the alcohol has at least 3 carbons and at least 2 hydroxyl groups. The preferred embodiment of the '140 patent is to use potassium hydroxide in propylene glycol. The use of solvent in the conjugation (isomerization) step gives rise to the potential formation of unwanted CLA-alcohol esters (e.g. CLA-propylene glycol esters).
U.S. Pat. No. 3,162,658 (the '658 patent) describes the use of alkali metal hydrocarbyl alcoholates or alkali metal amides to isomerize esters of unconjugated polyethylene acids such as linoleic acids. But it uses polar solvents for the isomerization step, which is undesirable. And the '658 patent also makes no mention of converting the resultant conjugated esters to the corresponding acids.
U.S. Pat. No. 3,984,444 (Ritz, et al., the '444 patent) describes the isomerization of an ester of an alcohol having 1 to 12 carbon atoms and an fatty acid having 10 to 24 carbon atoms and isolated double bonds to the corresponding compound having conjugated double bonds using alkaline metal alcoholates in strongly polar aprotic solvents. As noted above, the use of solvents in the conjugation step is undesirable. The '444 patent does not teach how to convert the resultant conjugated esters to the corresponding acids as well.
Typical procedures for the conversion of fatty acid methyl esters (FAME) to fatty acids (FA), such as those described in U.S. Pat. No. 4,185,027 and U.S. Pat. No. 5,872,289, involve the use of acidic catalysts. The use of such acidic catalysts is undesirable.
WO 01/14304 uses steam in the presence of a catalyst to directly hydrolyze FAME to FA. The reaction is carried out at a heightened temperature, which leads to the formation of undesirable CLA isomers, including the trans, trans-CLA isomers. Similarly, WO 97/07187 uses near critical temperatures and supercritical pressures to accomplish the transformation of FAME to FA.
GB 1589314 uses alkali metal hydroxides in alkyl nitrile solution for the conversion of FAME to FA.
As previously alluded to, CLAs have a wide variety of nutritional, therapeutic, and pharmacological uses. These uses include for example, body fat reduction, body weight reduction, increased muscle mass, increased feed efficiency, attenuated allergic reactions, prevention of weight loss due to immune stimulation, elevated CD-4 and CD-8 cells counts in animals, increased bone mineral content, prevention of skeletal abnormalities in animals and decreased blood cholesterol levels. The anticarcinogenic properties of CLA have been well documented. Administration of CLA inhibits rat mammary tumorigenesis, as demonstrated by Ha, et al., Cancer Res., 52: 2035s (1992). Ha, et al., Cancer Res., 50: 1097 (1990) reported similar results in a mouse forestomach neoplasia model. CLA has also been identified as a strong cytotoxic agent against target human melanoma, colorectal and breast cancer cells in vitro. A recent major review article confirms the conclusions drawn from individual studies. See Ip, Am. J. Clin. Nutr., 66 (6 Supp): 1523s (1997).
More recently, much attention has focused on CLA nutritively as a dietary supplement. CLA has been found to exert a profound generalized effect on body composition, in particular redirecting the partitioning of fat and lean tissue mass. See, e.g., U.S. Pat. No. 5,554,646 (Cook, et al.), which discloses a method utilizing CLA as a dietary supplement in various mammals, wherein a significant drop in fat content was observed with a concomitant increase in protein mass. See also, U.S. Pat. No. 5,428,072 (Cook, et al.), which disclosed that incorporation of CLA into animal feed (birds and mammals) increased the efficiency of feed conversion leading to greater weight gain in the CLA supplemented animals; the potential beneficial effects of CLA supplementation for food animal growers is apparent.
CLA is naturally occurring in foods and feeds consumed by humans and animals alike. In particular, CLA is abundant in products from ruminants. For example, several studies have been conducted in which CLA has been surveyed in various dairy products. Aneja, et al., J. Dairy Sci., 43: 231 (1990) observed that processing of milk into yogurt resulted in a concentration of CLA. Linoleic acid is an important component of biolipids, and comprises a significant proportion of triglycerides and phospholipids. Linoleic acid is known as an “essential” fatty acid, meaning that the animal must obtain it from exogenous dietary sources since it cannot be autosynthesized.
U.S. Pat No. 6,203,843 and U.S. Pat. No. 6,042,869 (both to Remmereit, J.) disclose bulk animal feeds containing CLA. U.S. Pat. No. 6,242,621 (Jerome et. al.) and U.S. Pat. No. 6,333,353 (Saebo, et al.) both disclose isomer enriched CLA compositions and methods of preparing such compositions.
The problem with most CLA products made by conventional approaches is their heterogeneity, and substantial variation in isoform from batch to batch. Considerable attention has been given to the fact that the ingestion of large amounts of hydrogenated oils and shortenings, instead of animal tallow, has resulted in a diet high in trans-fatty acid content For example, Holman, et al., PNAS, 88:4830 (1991) showed that rats fed hydrogenated oils gave rise to an accumulation in rat liver of unusual polyunsaturated fatty acid isomers, which appeared to interfere with the normal metabolism of naturally occurring polyunsaturated fatty acids. These concerns were summarized in an early Editorial in Am. J. Public Health, 84: 722 (1974).
Therefore, there exists a strong need for an improved process to produce a superior CLA composition, which is enriched with highly desired cis-9, trans-11- and trans-10, cis-12-CLA isomers, but which is low in certain undesirable CLA isomers and unwanted ester side products. Additionally, there is a need for an improved process to readily and economically prepare such CLA compositions in a safer and more environmental friendly way.